There is increasing appreciation that B cells and antibodies are important players in the immunopathogenesis of autoimmune demyelinating disorders of the central nervous system (CNS) such as neuromyelitis optica (NMO) and multiple sclerosis (MS). NMO is a severe inflammatory demyelinating disease affecting the optic nerves and spinal cord. Aquaporin-4 (AQP4) has been identified as a target of pathogenic autoantibody responses in NMO. Thus, NMO can be considered a model disease for CNS directed B cell and antibody autoimmunity. AQP4 is a water channel protein, which is expressed in astrocytes of the CNS. Anti- AQP4 antibodies have been proposed to exert neuropathological effects by a number of different mechanisms in NMO, including complement activation, antibody-dependent cell-mediated cytotoxicity, and downregulation of AQP4 and associated proteins. However, the exact epitope specificities of pathogenic anti-AQP4 antibodies remain unknown. It is unclear whether antigens other than AQP4 are also targets of pathologically relevant autoantibodies and no information is available regarding epitope targets of pathogenic autoantibodies in anti- AQP4 seronegative patients. However, there is evidence for extensive B cell-dependent autoimmunity in NMO, which likely translates into a diverse, pathogenic, CNS-directed autoantibody repertoire. We hypothesize that the AQP4-specific antibody repertoire is diverse, both in regards to AQP4 epitope recognition and with respect to pathological effects on CNS tissue. To address this hypothesis, we will first delineate the repertoire of AQP4 epitopes recognized by a.) recombinant monoclonal antibodies (rAbs) generated from CSF plasma cells, and by b.) purified soluble immunoglobulins from serum and CSF from anti- AQP4 seropositive NMO patients. Subsequently, monoclonal anti-AQP4 antibodies representative of individual AQP4-epitopes will be further characterized regarding their potential to damage AQP4 expressing cells. To establish an in vitro experimental system as close to in vivo as possible, we will also study pathological effects of anti-AQP4 antibodies on astrocytes derived from induced pluripotent stem cells (iPSC) from NMO patients. We also hypothesize, that antibody mediated immunity against targets other than AQP4 may play a role in AQP4-seronegative and possibly even AQP4-seropositive NMO patients. To address this hypothesis, we propose to identify targets of AQP4 non-reactive antibodies from AQP4-seronegative and -seropositive NMO patients. To achieve this goal, we will determine the reactivity of AQP4 non-reactive antibodies with iPSC-derived CNS cells (e.g. astrocytes, oligodendrocytes or neurons) and study pathological effects resulting from this autoantibody binding. These studies will elucidate pathologically relevant antibody-antigen interactions and are expected to serve as an important foundation for the development of disease-specific therapies and novel biomarkers.